FXR receptor-mediated modulation of cholesterol metabolism

ABSTRACT

This invention provides methods and compositions that are useful for modulating cholesterol levels in a cell, and for identifying compounds that can tested for ability to modulate cholesterol levels in mammals. In vitro assays for prescreening to identify candidate therapeutic agents for modulation of cholesterol metabolism are provided. These methods involve analyzing the effect of a test compound on the binding of FXR to a ligand for FXR. Such ligands include, for example, bile acids, coactivators, and corepressors. The methods and compositions involve modulating FXR-mediated expression of genes involved in cholesterol metabolism.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional PatentApplication No. 60/115,249, which was filed on Jan. 7, 1999, whichapplication is incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods and compounds for themodulation of cholesterol metabolism in a mammal and methods forscreening to identify candidate therapeutic agents for modulation ofcholesterol levels.

[0004] 2. Background

[0005] Atherosclerosis is a leading cause of death, myocardialinfarctions, stroke, peripheral vascular disease and cardiovasculardisease (Libby, in Chapter 242 of Harrison 's Principles of InternalMedicine, 14th edition (1998) (Fauci et al., eds.); Witztum, in Chapter36 of Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thedition (1996) (Hardman et al., eds.)). One of the major contributingfactors to atherosclerosis is hypercholesterolemia. Hypercholesterolemiais currently treated with a combination of dietary and pharmaceuticaltherapies. Often more than a single pharmaceutical agent and a dietaryregimen are necessary to decrease total cholesterol and LDL cholesterollevels to the desired level. Drugs such as bile acid sequestrants,niacin and the statins are commonly used to treat hypercholesterolemiaand atherosclerosis. The use of niacin, however, is limited by the highincidence (>50%) of numerous side effects that are experienced inpatients. Thus, a need for therapeutic agents that would decreasecholesterol levels still exists.

[0006] Cholesterol homeostasis in mammals is maintained through thecoordinate regulation of three major pathways in the liver. Two pathwayssupply cholesterol to cells and include an endogenous biosyntheticpathway in which acetate is converted into cholesterol and an exogenouspathway in which members of the low-density lipoprotein receptor familybind and internalize cholesterol-carrying particles from the blood. Athird pathway involves the conversion of cholesterol into hydrophilicbile acids.

[0007] The conversion of cholesterol to bile acids involves a minimum offourteen enzymes of the bile acid biosynthetic pathway. The first andrate-limiting enzymatic reaction of this pathway is catalyzed by theenzyme cholesterol 7α-hydrolase. The cholesterol 7α-hydrolase gene, alsoknown as cyp7A, belong to the cytochrome P-450 family that contains manymicrosomal enzymes involved in liver metabolism. It has been shown thatexpression of the cyp7A gene is tightly regulated. This gene isexpressed exclusively in the liver and its expression can be induced bydietary cholesterol and suppressed by bile acids. An alternative pathwayinvolves the cyp7B gene, which is expressed in the brain as well asother tissues. It has been shown that cholesterol catabolism plays acentral role in cholesterol homeostasis. For instance, treatment oflaboratory animals with cholestid or cholestyramine, two bileacid-binding resins, decreases serum cholesterol levels. Moreover,overexpression of the cyp7 gene in hamsters reduces both totalcholesterol levels and LDL cholesterol levels. As such, cholesterol7α-hydrolase is a potential therapeutic target for the identification ofcholesterol reducing compounds and, thus, understanding the mechanism bywhich expression of the cyp7 genes are regulated is of particularimportance.

[0008] Nuclear receptors form a large family of ligand-activatedtranscription factors that modify the expression of target genes bybinding to specific cis-acting sequences (Laudet et al. (1992) EMBO J.11: 1003-1013; Lopes da Silva et al. (1995) Trends Neurosci. 18:542-548; Mangelsdorf et al. (1995) Cell 83: 835-839; Mangelsdorf andEvans (1995) Cell 83: 841-850). Nuclear receptors include those thatremain sequestered in the cytoplasm in the absence of their cognateligands (e.g., steroid hormone receptors). Upon binding of the ligand,the steroid hormone receptors are translocated to the nucleus where theybind to hormone response elements, typically as homodimers.

[0009] Most of the nuclear receptors, conversely, are not sequestered inthe cytoplasm in the absence of their ligands but rather remain in thenucleus. These receptors, which include the thyroid hormone, retinoid,fatty acid, and eicosanoid receptors, typically bind to their cognateresponse elements as heterodimers with a 9-cis-retinoic acid receptor(RXR). Often, binding of a nuclear receptor to a response element occursin the absence of the cognate ligand.

[0010] One such nuclear receptor is the famesoid X receptor (FXR; alsoknown as “RIP 14” and “NR1H4”) (Forman et al. (1995) Cell 81: 687; Seolet al. (1995) Mol. Endocrinol. 9: 72; Mangelsdorf and Evans, supra.).FXR functions as a heterodimer with RXR, and several isoprenoid lipidscan weakly activate FXR at supraphysiological concentration; however,these compounds do not activate all species of FXR and do not bind asligands (Forman et al., supra.; Zavacki et al. (1997) Proc. Nat'l. Acad.Sci. USA 94: 7909). Thus, the identity and physiologic function of FXRligands have remained unknown.

[0011] The lack of understanding of physiological mechanisms thatregulate cholesterol levels has hampered the discovery of improvedmethods of treating hypercholesteremia. Thus, a need exists forcharacterization of regulatory mechanisms of cholesterol hemostasis, andfor screening methods to identify better compounds for treatinghypercholesteremia. The present invention fulfills these and otherneeds.

SUMMARY OF THE INVENTION

[0012] The present invention is based in part on the discovery that theorphan nuclear receptor FXR (famesoid X receptor) is involved in thesuppression of the human cyp7 genes. More particularly, it has beendiscovered that FXR functions as a bile acid receptor/sensor thatmediates cyp7 expression in a bile-acid dependent manner. As such, inone aspect, the present invention provides a method for increasingcholesterol metabolism in a cell by contacting the cell with a compoundthat modulates the binding of an FXR to a ligand for FXR. In oneembodiment, the ligand is a bile acid. In another embodiment, thecompound modulates the binding of the FXR to an RXR or other coactivatoror corepressor.

[0013] The invention provides methods for prescreening to identify acandidate therapeutic compound suitable for testing for ability tomodulate cholesterol metabolism in a cell. These methods involve:

[0014] providing a reaction mixture that comprises:

[0015] a) a polypeptide that comprises a ligand binding domain of anFXR;

[0016] b) a ligand for FXR; and

[0017] c) a test compound; and

[0018] determining whether the amount of binding of the FXR ligandbinding domain to the ligand for FXR is increased or decreased in thepresence of the test compound compared to the amount of binding in theabsence of the test compound. A test compound that causes an increase ordecrease in binding is a candidate therapeutic agent for modulation ofcholesterol metabolism. In some embodiments, the methods further involveadministering the candidate therapeutic agent to a cell to determinewhether the candidate therapeutic agent modulates cholesterol metabolismin the cell.

[0019] The invention also provides methods for screening to identify acompound that modulates cholesterol metabolism in a cell using a geneexpression assay. For example, these methods can involve contacting acell with a test compound, wherein said cell includes:

[0020] a) a polynucleotide that encodes a polypeptide comprising: 1) aDNA binding domain of a receptor which binds to DNA; and 2) a ligandbinding domain that is substantially identical to a ligand bindingdomain of a FXR;

[0021] b) a ligand for FXR; and

[0022] c) a reporter gene construct which comprises a response elementto which said DNA binding domain can bind, wherein said response elementis operably linked to a promoter that is operative in the cell and saidpromoter is operably linked to a reporter gene.

[0023] By determining whether the reporter gene is expressed at a higheror lower level in the presence of said test compound compared to saidreporter gene expression level in the absence of said test compound, onecan identify a test compound that can modulate cholesterol metabolism ina cell. The cells used in the methods of the invention are typicallymammalian cells. In a presently preferred embodiment, the cell is in amammal and, in particular, in a human.

[0024] The test compounds are, in some embodiments, a compound thatbinds to FXR or to the ligand for FXR, and thus inhibits binding of theligand to the FXR. For example, the compound can be an antibody thatbinds to FXR, or an organic molecule that interferes with theinteraction. The binding of the compound can also modulate the bindingof a transcription complex that comprises FXR to a response element.Response elements of particular interest include those that are derivedfrom a region upstream of a cyp7 gene.

[0025] In another embodiment, the present invention provides a methodfor reducing cholesterol levels in a mammal. These methods involveadministering to said mammal a compound that modulates the binding of anFXR to an FXR response element. In a preferred embodiment, the mammal isa human.

[0026] The invention also provides assays for identifying ligands forFXR, and response elements that are bound by FXR or by a transcriptioncomplex that includes FXR.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIGS. 1A and 1B illustrate that the bile acid CDCA specificallytransactivates FXR. FIG. 1A illustrates that a GAL4 DNA binding domainfused to an FXR ligand binding domain is activated by CDCA, while ligandbinding domains of other nuclear receptors fused to the GAL4 DBD are notactivated by CDCA. FIG. 1B illustrates that CDCA-mediatedtransactivation of FXR is dose-dependent.

[0028]FIGS. 2A and 2B illustrate that FXR suppresses cyp7 expression.FIG. 2A illustrates that CDCA-mediated suppression of cyp7 is specificto FXR. FIG. 2B illustrates a dose-response profile of CDCA-mediatedsuppression of cyp7 expression by FXR.

[0029]FIGS. 3A, 3B and 3C illustrate that FXR suppression is specific tocyp7. FIG. 3A illustrates that FXR does not suppress expression of thelow density lipoprotein (LDL) receptor. FIG. 3B illustrates thattitrating away FXR causes a loss of cyp7 suppression. FIG. 3Cillustrates that FXR mutants do not suppress cyp7 expression.

[0030]FIGS. 4A and 4B, 4C and 4D illustrate the effect of different bileacids on FXR activity. FIG. 4A illustrates a western blot analysis ofcyp7 expression, as well as a quantitative RT-PCR analysis of cyp7 mRNAin HepG2 cells. FIG. 4B illustrates that FXR interacts with theco-activator SRC-1 in a mammalian two-hybrid assay. FIG. 4C shows theeffect of different bile acids on FXR-mediated transactivation. FIG. 4Dshows the effect of the bile acids on FXR-mediated suppression of cyp7expression.

[0031]FIG. 5A illustrates the results of a peptide sensor assay using aGST-FXR fusion protein. The binding of the peptide sensor to the FXR LBDin the presence of different bile acids and derivatives was analyzed byfluorescence polarization. FIG. 5B shows the results of an analysis inwhich FRET was used to monitor the effect of bile acids and derivativeson FXR/SRC-1 interaction. FIG. 5C shows the results of a sensor peptidestudy in which ELISA was used to detect binding of the sensor peptide tothe FXR LBD in the presence of increasing concentrations of differentbile acids and derivatives.

DETAILED DESCRIPTION

[0032] The following definitions are used herein.

[0033] Definitions

[0034] The term “isolated” refers to material that is substantially oressentially free from components which normally accompany the enzyme asfound in its native state. Thus, the polypeptides of the invention donot include materials normally associated with their in situenvironment. Typically, isolated proteins of the invention are at leastabout 80% pure, usually at least about 90%, and preferably at leastabout 95% pure as measured by band intensity on a silver stained gel orother method for determining purity. Protein purity or homogeneity canbe indicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualization upon staining. For certain purposes high resolution willbe needed and HPLC or a similar means for purification utilized.

[0035] The term “recombinant” when used with reference to a cell, ornucleic acid, or vector, indicates that the cell, or nucleic acid, orvector, has been modified by the introduction of a heterologous nucleicacid or the alteration of a native nucleic acid, or that the cell isderived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all.

[0036] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual alignmentand inspection.

[0037] The phrase “substantially identical,” in the context of twonucleic acids or polypeptides, refers to two or more sequences orsubsequences that have at least 75%, preferably 85%, most preferably90-95% nucleotide or amino acid residue identity, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection.Preferably, the substantial identity exists over a region of thesequences that is at least about 50 residues in length, more preferablyover a region of at least about 100 residues, and most preferably thesequences are substantially identical over at least about 150 residues.In a most preferred embodiment, the sequences are substantiallyidentical over the entire length of the coding regions and/oruntranslated regions.

[0038] A further indication that two nucleic acid sequences orpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

[0039] “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

[0040] The phrase “hybridizing specifically to”, refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridize toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm, 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na⁺, typically about 0.01 to 1.0M Na⁺ concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

[0041] The phrase “specifically or selectively binds to an antibody” or“specifically immunoreactive with”, when referring to a protein orpeptide, refers to a binding reaction which is determinative of thepresence of the protein in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated assay conditions,the specified antibodies or other ligand bind to a particular proteinand do not bind in a significant amount to other proteins present in thesample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, antibodies raised to the FXRpolypeptides (or subsequences thereof) or to the polypeptides partiallyencoded by the FXR polynucleotide sequences can be selected to obtainantibodies specifically immunoreactive with the full length proteins andnot with other proteins, except perhaps to polymorphic variants. Avariety of immunoassay formats can be used to select antibodies andother molecules that specifically bind to a particular protein such asFXR. For example, solid-phase ELISA immunoassays are routinely used toselect monoclonal antibodies specifically immunoreactive with a protein.See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention pertains to the discovery that ligands forthe formerly orphan receptor FXR include bile acids, and that thebinding of a bile acid to FXR results in suppression of cholesterolcatabolism. Accordingly, the invention provides methods for modulating(i.e., increasing or decreasing) cholesterol levels in a cell bycontacting the cell with a compound that modulates the binding of FXR toligand for FXR. By decreasing binding of the FXR to an FXR ligand, forexample, a compound can reduce the suppression of a gene such as cyp7(i.e., increase the expression) which encodes an enzyme that is a keyenzyme in cholesterol metabolism. Also provided by the invention aremethods for increasing or decreasing cholesterol levels in a mammal,including humans by administering to the mammal a compound thatmodulates the binding of FXR to an FXR ligand. In other embodiments, theinvention provides methods for identifying compounds that can modulatecholesterol metabolism by, for example, altering the ability of FXR tobind to FXR ligands.

[0043] A. Assays for Identifying Compounds that Modulate CholesterolMetabolism

[0044] The invention also provides screening assays for identifyingcompounds that can modulate cholesterol metabolism. These compounds canfunction by, for example, altering the interaction between FXR and FXRligands (e.g., bile acids, corepressors and/or coactivators) and/orbetween FXR and its response elements. Of particular interest is themodulation of cyp7 gene expression, which is inhibited by binding of FXRto the upstream region of the gene. Thus, a compound that inhibits thecis-repressing activity of FXR can increase the expression of cyp7, thusincreasing degradation of cholesterol. Compounds that are identifiedusing the screening methods of the invention find use in studies of generegulation, and also find therapeutic use in situations in which it isdesirable to increase or decrease cholesterol metabolism. Other useswill also be apparent those of ordinary skill in the art.

[0045] The assays of the invention are amenable to screening of largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). Essentially any chemical compound can be used as a potentialFXR activity modulator in the assays of the invention. In someembodiments, compounds that can be dissolved in aqueous or organic(especially DMSO-based) solutions are used. It will be appreciated thatthere are many suppliers of chemical compounds, including Sigma (St.Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0046] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial library containing a large number ofpotential therapeutic compounds (potential modulator compounds). Such“combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity (e.g., inhibit the interaction between FXR andan FXR ligand). The compounds thus identified can serve as conventional“lead compounds” or “candidate therapeutic agents,” or can themselves beused as potential or actual therapeutics.

[0047] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0048] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (PCT PublicationNo. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S.Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with β-D-glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel,Berger and Sambrook, all supra), peptide nucleic acid libraries (see,e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaugluiet al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries(see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

[0049] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Wobum, Mass., 433A Applied Biosystems,Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

[0050] The invention provides both biochemical and cell-based assays foridentifying compounds that can modulate FXR-mediated regulation ofcholesterol metabolism. Often, an initial assay is performed in vitro toidentify compounds that are potential candidate therapeutic agents,after which such compounds are then tested in vivo by, for example,administering the compound to a test animal to determine whethercholesterol levels are affected.

[0051] 1. Direct and Displacement Assays

[0052] The invention provides methods for identifying compounds that aresuitable for further testing as candidate therapeutic agents fortreatment of hypercholesteremia and other conditions that are associatedwith cholesterol synthesis and metabolism. Compounds that exhibit thedesired activity in the in vitro assays can be used for further studiesof the regulation of pathways involved in cholesterol hemostasis, or canbe subjected for further testing to identify those that are suitable foruse to treat hypercholesterolemia or other conditions involvingcholesterol metabolism.

[0053] One type of assay that can be used is a direct binding assay,which measures the amount of a compound that can bind to an FXRpolypeptide or to a polypeptide that has an FXR ligand binding domain.These assays can be carried out using labeled candidate therapeuticagents which are then incubated with a polypeptide that has an FXRligand binding domain (e.g., a full-length FXR polypeptide or a fusionprotein). Labels include radioisotopes, immunochemicals, fluorophores,and the like. Those of skill in the art will recognize a variety of waysof separating the bound labeled candidate therapeutic agent from thefree labeled candidate therapeutic agent. The affinity of the labeledcandidate therapeutic agent for an FXR polypeptide can be calculatedusing standard ligand binding methods.

[0054] Another type of assay that can be used to pre-screen candidatetherapeutic agents involves testing the ability of a test compound tomodulate binding of FXR to a ligand for FXR. These can be conducted, forexample, as a direct binding assay with a labeled FXR ligand in thepresence of a candidate therapeutic agent. The assays involve placingthe test compound into an assay mixture that includes at least a ligandbinding domain of an FXR polypeptide and a ligand for FXR. The effect onbinding of the FXR ligand to FXR is determined. A test compound thatdecreases the amount of labeled FXR ligand that is bound to an FXRpolypeptide or a polypeptide that has an FXR ligand binding domain, isof interest for future screening for its ability to modulate cholesterollevels in vivo.

[0055] Ligands that are suitable for use in the assays of the inventioninclude, but are not limited to, bile acids and related compounds suchas CDCA (chenodeoxycholic acid), GCDCA (glycochenodeoxycholic acid),TCDCA (taurochenodeoxycholic acid), GCA (glycocholic acid), TCA(taurocholic acid), DCA (deoxycholic acid), LCA (lithocholic acid), DHCA(dehydrocholic acid), UDCA (ursodeoxycholic acid) and CA (cholic acid).In a presently preferred embodiment, the bile acid is CDCA. Additionalbile acids and other ligands are described in, for example, Makishima etal. (1999) Science 284: 1362-1365. The assays can also employcoactivators and corepressors with which FXR interacts. Methods ofidentifying FXR ligands are described below.

[0056] In presently preferred embodiments, an assay such as thefluorescence polarization assay or the fluorescence resonance energytransfer assay is employed to identify candidate therapeutic agents.These assays do not require the separation of bound and free labeledtest compound. Fluorescence polarization (FP) or fluorescence anisotropyis a useful tool for the study of molecular interactions (see, e.g.http://www.panvera.com/tech/appguide/fpintro.html, Nov. 4, 1999). First,a molecule labeled with a fluorophore is excited with plane polarizedlight. If the fluorescent molecule stays stationary while in the excitedstate, light is emitted in the same polarized plane. If the excitedfluorescently labeled molecule rotates out of the plane of the polarizedlight while in the excited state, light is emitted from the molecule ina different plane. For example, if vertical polarized light is used toexcite the fluorophore, the emission spectra can be monitored in thevertical and horizontal planes. Fluorescence polarization is calculatedas shown in the following Formula I:

Fluorescent polarization=P=(Int ∥−Int^(⊥))/(Int ∥+Int^(⊥))  I

[0057] In Formula I, Int ∥ is the intensity of the emission parallel tothe excitation plane. Int^(⊥) is the intensity of the emissionperpendicular to the excitation plane.

[0058] A small fluoreseently labeled molecule, when free in solution,can emit depolarized light when excited with the proper wavelength oflight. If, however, the molecule (e.g., a ligand) binds to a secondmolecule (e.g., a receptor) the fluorescently labeled molecule is moreconstrained so the light emitted is more polarized and the fluorescencepolarization (FP) value is higher. Thus, a higher FP value indicatesthat the fluorescently labeled molecule is able to bind to the secondmolecule. A competition assay also can be performed using FP. If anunlabeled molecule is present in the solution, then it will compete forbinding to the second molecule, e.g., the antibody and the FP value willbe decreased. Thus, FP can be used in competitive assays.

[0059] Commercial assays exist to test the affinity of compounds forhuman estrogen receptor α and β using a fluorescently labeled estrogencompound (see, Panvera, (Madison, Wis.) publications Lit.#'s L0069,L0082, L0084, L0095, L0072, L0085). Similarly, test compounds can befluorescently labeled with a fluorophore that is active in a FP assay.For example, N-terminal amines of proteins, peptide, or peptide analogscan be labeled with fluorescein (Panvera, publications Lit. # L0057 andL0059) or a small fluorescent compound. Briefly, afluorescein-C₆-succinimidyl ester can be conjugated to peptides orproteins. The fluorescein labeled peptide/protein can then be purifiedfrom the unreacted fluorescein-C6-succinimidyl ester using thin-layerchromatography or gel filtration chromatography. If the labeled testcompound can bind to a polypeptide that has an FXR ligand bindingdomain, the level of polarization is increased. The FP assay also can beused to assay the ability of a fluorescently labeled FXR ligand to bindto an FXR polypeptide.

[0060] Alternatively, a test compound can be screened for its ability todecrease the FP of a fluorescently labeled known FXR ligand complexedwith an FXR polypeptide or a polypeptide comprising an FXR ligandbinding domain. Briefly, a known FXR ligand is labeled with afluorescent moiety. A test compound that decreases the FP value of thefluorescently labeled FXR ligand and FXR is displacing or inhibiting theability of the fluorescently labeled FXR ligand to bind to the ligandbinding domain of FXR.

[0061] Methods employing the technique of fluorescence resonance energytransfer (FRET) can be employed using the methods and compositions ofthe present invention. FRET occurs between two fluorophores when theexcitation of the donor fluorophore is transferred to the acceptorfluorophore. This interaction is dependent on the distance between thedonor and acceptor fluorophore and distance-dependent interactionbetween a donor and acceptor molecule. The donor and acceptor moleculesare fluorophores. If the fluorophores have excitation and emissionspectra that overlap, then in close proximity (typically around 10-100angstroms) the excitation of the donor fluorophore is transferred to theacceptor fluorophore. The relative proximity of the first and secondlabels is determined by measuring a change in the intrinsic fluorescenceof the first or second label. Commonly, the emission of the first labelis quenched by proximity of the second label.

[0062] Many appropriate interactive labels for FRET are known. Forexample, fluorescent labels, dyes, enzymatic labels, and antibody labelsare all appropriate. Examples of preferred interactive fluorescent labelpairs include terbium chelate and TRITC (tetrarhodamine isothiocyanate),europium cryptate and allophycocyanin and many others known to one ofskill. Similarly, two colorimetric labels can result in combinationsthat yield a third color, e.g., a blue emission in proximity to a yellowemission produces an observed green emission.

[0063] With regard to preferred fluorescent pairs, there are a number offluorophores which are known to quench each other. Fluorescencequenching is a bimolecular process that reduces the fluorescence quantumyield, typically without changing the fluorescence emission spectrum.Quenching can result from transient excited interactions, (collisionalquenching) or, e.g., from the formation of nonfluorescent ground statespecies. Self-quenching is the quenching of one fluorophore by another;it tends to occur when high concentrations, labeling densities, orproximity of labels occurs. Some excited fluorophores interact to formexcimers, which are excited state dimers that exhibit altered emissionspectra (e.g., phospholipid analogs with pyrene sn-2 acyl chains); SeeHaugland (1996) Handbook of Fluorescent Probes and Research Chemicals,published by Molecular Probes, Inc., Eugene, Oreg.

[0064] The Forster radius (R_(o)) is the distance between fluorescentpairs at which energy transfer is 50% efficient (i.e., at which 50% ofexcited donors are deactivated by FRET). The magnitude of R_(o) isdependent on the spectral properties of donor and acceptor dyes:R_(o)=[8.8×10²³·K²·n⁻⁴·QY_(D)·J(λ)]^(1/6) Å; where K²=dipole orientationrange factor (range 0 to 4, K²=⅔ for randomly oriented donors andacceptors).; QY_(D)=fluorescence quantum yield of the donor in theabsence of the acceptor; n=refractive index; and J( )=spectral overlapintegral=∫ε_(A)(λ)·F_(D)·(λ4)dλ cm³M⁻¹, where ε_(A)=extinctioncoefficient of acceptor and FD=fluorescence emission intensity of donoras a fraction of total integrated intensity. Some typical R_(o) arelisted for typical donor acceptor pairs in Table 1: TABLE 1 DonorAcceptor R_(o) (Å) Fluorescein Tetramethylrhodamine 55 IAEDANSFluorescein 46 EDANS DABCYL 33 BODIPY FL BODIPY FL 57 Fluorescein QSY-7dye 61

[0065] An extensive compilation of R_(o) values are found in theliterature; see Haugland (1996), supra. In most uses, the donor andacceptor dyes are different, in which case FRET can be detected by theappearance of sensitized fluorescence of the acceptor or by quenching ofthe donor fluorescence. When the donor and acceptor are the same, FRETis detected by the resulting fluorescence depolarization.

[0066] In addition to quenching between fluorophores, individualfluorophores are also quenched by nitroxide-labeled molecules such asfatty acids. Spin labels such as nitroxides are also useful in theliquid phase assays of the invention.

[0067] Test compounds and a polypeptide that includes an FXR ligandbinding domain can be labeled with FRET pairs. If the test compound candirectly interact with the FXR ligand binding domain, fluorescenceresonance energy transfer can take place and the affinity can bemeasured. Alternatively, a known FXR ligand can be labeled with anappropriate FRET label and incubated with an FRET fluorophore labeledpolypeptide that includes an FXR ligand binding domain. Fluorescenceresonance energy transfer can take place between the labeled FXR ligandand the labeled FXR ligand binding domain. If a test compound wereincubated with the two labeled components, the amount of FRET would belowered if the test compound can inhibit or displace the binding of thelabeled FXR ligand to the FXR ligand binding domain.

[0068] Additional methods for assaying the ability of test compounds tomodulate FXR interactions with its ligands employ peptide sensors. Theseassays can be adapted from those described in WO 99/27365. Briefly,these assays use a peptide sensor to which is attached a detectablelabel. The peptides are based on corepressor or coactivator proteinmotif sequences, either naturally occurring or derived from mutationalanalysis. The peptide sensors are derived from corepressors orcoactivators that interact with FXR (e.g., a p160 coactivator, RXR, CPF,and others that are identified as described herein). Alternatively, thepeptides can be obtained through randomizing residues and selecting forbinding to the FXR receptor polypeptide. Panels of predetermined orrandomized candidate sensors can be screened for receptor binding. ForFXR peptide sensor assays, an example of a suitable peptide sensor isderived from the receptor-interacting domain of the coactivator SRC-1.This domain has been mapped to a short motif with the amino acidsequence LXXLL, where L is leucine and X is any amino acid. Fragments ofSRC-1 or short synthetic peptides containing one LXXLL motif or morebind nuclear receptors in a ligand-dependent manner (Darimont et al.(1998) Genes Dev. 12: 3343; McInerney et al. (1998) Genes Dev. 12:3357).

[0069] In typical embodiments, the sensor peptides are labeled with adetectable label. The detectable labels can be primary labels (where thelabel comprises an element that is detected directly or that produces adirectly detectable element) or secondary labels (where the detectedlabel binds to a primary label, as is common in immunological labeling).An introduction to labels, labeling procedures and detection of labelsis found in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue published by Molecular Probes, Inc., Eugene,Oreg. Primary and secondary labels can include undetected elements aswell as detected elements. Useful primary and secondary labels in thepresent invention can include spectral labels such as fluorescent dyes(e.g., fluorescein and derivatives such as fluorescein isothiocyanate(FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red,tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like), radiolabels (e.g., 3H,125I, 35S, 14C, 32P, 33P, etc.), enzymes (e.g., horse radish peroxidase,alkaline phosphatase etc.), spectral colorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. The label may be coupled directly orindirectly to a component of the detection assay (e.g., the detectionreagent) according to methods well known in the art. As indicated above,a wide variety of labels may be used, with the choice of label dependingon sensitivity required, ease of conjugation with the compound,stability requirements, available instrumentation, and disposalprovisions. In a presently preferred embodiment, the detectable label isa fluorescent label, in which case fluorescence polarization detectionprovides a sensitive and efficient means of detecting whether thepeptide sensor is bound to the FXR receptor polypeptide. See, e.g.,Schindler et al. (1995) Immunity 2: 689-697).

[0070] The sensor peptide and the FXR polypeptide are incubated underconditions that are suitable for sensor binding to the receptorpolypeptide. In some embodiments, a candidate modulator of FXR bindingto a corepressor, coactivator or other ligand is included in thereaction mixture. If a candidate modulator increases or decreasesbinding of the sensor peptide to the FXR polypeptide, the candidatemodulator is a potential lead compound for blocking the FXR-mediatedeffect on transcription.

[0071] 2. Cell-Based Screening Methods

[0072] The present invention also provides cell-based methods forscreening to identify compounds that are suitable for use as cholesterollevel-mediating therapeutic agents, or that are suitable to furtherscreening compounds that exhibit activity in the polypeptide-basedassays described above. These methods provide an assay to determinewhether expression of a gene involved in cholesterol hemostasis isaffected by administration of the test compound.

[0073] In some embodiments, these screening methods of the invention usea cell that contains a polypeptide that has a ligand binding domain(LBD) which is at least substantially identical to that of an FXR. Thepolypeptide typically will also include a DNA binding domain (DBD). TheDBD can be substantially identical to that of an FXR (i.e., afull-length FXR is used), or to that of a receptor other than FXR forwhich the response element is known (e.g., GAL4, nuclear hormonereceptors, and the like). Examples of suitable chimeric polypeptides aredescribed in more detail below. Conveniently, the chimeric receptorpolypeptide is introduced into the cell by expression of apolynucleotide that encodes the receptor polypeptide. For example, anexpression vector that encodes the chimeric receptor can be introducedinto the cell that is to be used in the assay. Suitable FXR polypeptidesand chimeric receptors are described below.

[0074] The cells will also contain a response element that can be boundby the DNA binding domain. Response elements, including glucocorticoidresponse elements (GRE) and estrogen response elements (ERE), aredescribed in, for example, Jantzen et al. (1987) Cell 49: 29; Martinezet al. (1987) EMBO J. 6: 3719 and Burch et al. (1988) Mol. Cell. Biol.8: 1123. Many other response elements are known; a commonly usedresponse element is the GAL4 upstream activating sequence (UAS_(G))(Keegan et al. (1986) Science 14: 699-704), which is responsive tobinding by chimeric receptors that include the GAL4 DNA binding domain.

[0075] The response element that is bound by the DNA binding domain usedin the chimeric polypeptide is generally used in a reporter geneconstruct. In such constructs, the response element is operably linkedto a promoter that is active in the cell. In presently preferredembodiments, the promoter is operably linked to a reporter gene that,when expressed, produces a readily detectable product. The responseelement/reporter gene construct is conveniently introduced into cells aspart of a “reporter plasmid.”

[0076] Suitable promoters include those described above. In presentlypreferred embodiments, the promoter is operably linked to a reportergene that, when expressed, produces a readily detectable product. Avariety of reporter gene plasmid systems are known, such as thechloramphenicol acetyltransferase (CAT) and β-galactosidase (e.g.,bacterial lacZ gene) reporter systems, the firefly luciferase gene (See,e.g., Cara et al. (1996) J. Biol. Chem., 271: 5393-5397), the greenfluorescence protein (see, e.g., Chalfie et al. (1994) Science 263:802)and many others. Examples of reporter plasmids are also described inU.S. Pat. No. 5,071,773. Selectable markers which facilitate cloning ofthe vectors of the invention are optionally included. Sambrook andAusubel, both supra, provide an overview of selectable markers.

[0077] In some embodiments of this assay, the reporter plasmid and anexpression plasmid that directs expression of the chimeric receptor areintroduced into a suitable host cell. Standard transfection methods canbe used to introduce the vectors into the host cells. For mammalian hostcells, preferred transfection methods include, for example, calciumphosphate precipitation (Chen and Okayama (1988) BioTechniques 6: 632),DEAE-dextran, and cationic lipid-mediated transfection (e.g.,Lipofectin) (see, e.g., Ausubel, supra.). In some cases, the host cell,prior to introduction of the expression plasmid, should not contain anFXR receptor. See, e.g., U.S. Pat. No. 5,071,773 for suitable host cellsfor use in the assays.

[0078] The assay methods involve contacting test cells that contain thereporter plasmid, the native or chimeric FXR polypeptide, and a ligand(e.g., bile acid) with the test compound. In some embodiments, an RXRpolypeptide and/or other coactivators and corepressors which mediate theeffect of FXR are also present in the test cells. For example, a cellthat contains a reporter gene construct and the chimeric peptide can begrown in the presence and absence of putative modulatory compounds.

[0079] The observed effect on reporter gene expression can depend on theparticular assay system used. For example, when an FXR polypeptide thatincludes the FXR DBD, AF-2 domain, and LBD is used, cells grown in theabsence of the FXR ligand will exhibit a level of reporter geneexpression that is above the level observed in the absence of theligand. Conversely, when a GAL4 DBD is used, binding of the ligand tothe fusion polypeptide will result in increased expression the reportergene to which is linked the GAL4 response element. Therefore, reportergene expression is increased when cells are grown in the presence of aligand for the FXR.

[0080] B. FXR Polypeptides and Fusion Polypeptides

[0081] The assays of the invention typically employ an FXR polypeptide.The FXR polypeptide can be a full-length FXR, or can include one or moredomains of FXR. In some embodiments, one or more FXR domains (e.g., aDNA binding domain (DBD) or a ligand binding domain (LBD)) are used as afusion protein with a domain from another polypeptide, such as anotherreceptor. For example, some assay formats use a fusion protein thatincludes an FXR LBD fused to a DBD of another receptor.

[0082] The FXR polypeptides and fusion polypeptides used in the assaysof the invention can be made by methods known to those of skill in theart. For example, the FXR proteins or subsequences thereof can besynthesized using recombinant DNA methodology. Generally this involvescreating a DNA sequence that encodes the polypeptide, modified asdesired, placing the DNA in an expression cassette under the control ofa particular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

[0083] FXR polypeptides, and polynucleotides that encode FXRpolypeptides, are known to those of skill in the art. For example, cDNAsequences of FXR polypeptides from human (U68233), Branchiostomalanceolatum (U93409), Danio rerio (zebrafish) (U93467), and rat (U18374)are found in GenBank. The nucleic acids that encode FXR can be used toexpress the FXR polypeptide, or to construct genes that encode a desiredfusion polypeptide.

[0084] FXR-encoding nucleic acids can be isolated by cloning oramplification by in vitro methods, such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (SSR). A wide variety of cloning and in vitro amplificationmethodologies are well-known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger, Sambrook, andAusubel (all supra.); Cashion et al., U.S. Pat. No. 5,017,478; and Carr,European Patent No. 0,246,864. Examples of techniques sufficient todirect persons of skill through in vitro amplification methods are foundin Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S.Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications(Innis et al., eds) Academic Press Inc. San Diego, Calif. (1990)(Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal OfNIH Research (1991) 3: 81-94; (Kwoh et al. (1989) Proc. Nat'l Acad. Sci.USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826; Landegren et al.(1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene, 4: 560; and Barringer et al. (1990)Gene 89: 117.

[0085] In one preferred embodiment, FXR cDNAs can be isolated by routinecloning methods. The cDNA sequence provided in GenBank, for example, canbe used to provide probes that specifically hybridize to a FXR gene in agenomic DNA sample, to an FXR mRNA in a total RNA sample, or to a FXRcDNA in a cDNA library (e.g., in a Southern or Northern blot). Once thetarget FXR nucleic acid is identified, it can be isolated according tostandard methods known to those of skill in the art (see, e.g., Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook etal.); Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)(Ausubel). In another preferred embodiment, the FXR nucleic acids can beisolated by amplification methods such as polymerase chain reaction(PCR).

[0086] A polynucleotide that encodes an FXR polypeptide or fusionprotein can be operably linked to appropriate expression controlsequences for a particular host cell in which the polypeptide is to beexpressed. For E. coli, appropriate control sequences include a promotersuch as the T7, trp, or lambda promoters, a ribosome binding site andpreferably a transcription termination signal. For eukaryotic cells, thecontrol sequences typically include a promoter which optionally includesan enhancer derived from immunoglobulin genes, SV40, cytomegalovirus,etc., and a polyadenylation sequence, and may include splice donor andacceptor sequences. In yeast, convenient promoters include GAL1,10(Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russellet al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982)6:675-680), and MFα1 (Herskowitz and Oshima (1982) in The MolecularBiology of the Yeast Saccharomyces (eds. Strathem, Jones, and Broach)Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).

[0087] Expression cassettes are typically introduced into a vector whichfacilitates entry into a host cell, and maintenance of the expressioncassette in the host cell. Vectors that include a polynucleotide thatencodes an FXR polypeptide are provided by the invention. Such vectorsoften include an expression cassette that can drive expression of theFXR polypeptide. To easily obtain a vector of the invention, one canclone a polynucleotide that encodes the FXR polypeptide into acommercially or commonly available vector. A variety of common vectorssuitable for this purpose are well known in the art. For cloning inbacteria, common vectors include pBR322 derived vectors such aspBLUESCRIPT™, and k-phage derived vectors. In yeast, vectors includeYeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids(the YRp series plasmids) and pGPD-2. A multicopy plasmid with selectivemarkers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used. Anumber of yeast expression plasmids such as YEp6, YEp13, YEp4 can beused as expression vectors. The above-mentioned plasmids have been fullydescribed in the literature (Botstein et al. (1979) Gene 8:17-24; Broachet al. (1979) Gene, 8:121-133). For a discussion of yeast expressionplasmids, see, e.g., Parents, B., YEAST (1985), and Ausubel, Sambrook,and Berger, all supra). Expression in mammalian cells can be achievedusing a variety of commonly available plasmids!, including pSV2,pBC12BI, and p91023, as well as lytic virus vectors (e.g., vacciniavirus, adenovirus, and baculovirus), episomal virus vectors (e.g.,bovine papillomavirus), and retroviral vectors (e.g., murineretroviruses).

[0088] FXR polypeptides and fusion polypeptides that include at leastone FXR domain can be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeasts, filamentous fungi, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma cell lines. Techniques for gene expression in microorganisms aredescribed in, for example, Smith, Gene Expression in RecombinantMicroorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994.Examples of bacteria that are useful for expression include, but are notlimited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus. Filamentous fungi that areuseful as expression hosts include, for example, the following genera:Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium,Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia. See, e.g., U.S.Pat. No. 5,679,543 and Stahl and Tudzynski, Eds., Molecular Biology inFilamentous Fungi, John Wiley & Sons, 1992. Synthesis of heterologousproteins in yeast is well known and described in the literature. Methodsin Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory,(1982) is a well recognized work describing the various methodsavailable to produce the enzymes in yeast.

[0089] The nucleic acids that encode the polypeptides of the inventioncan be transferred into the chosen host cell by well-known methods suchas calcium chloride transformation for E. coli and calcium phosphatetreatment or electroporation for mammalian cells. Cells transformed bythe plasmids can be selected by resistance to antibiotics conferred bygenes contained on the plasmids, such as the amp, gpt, neo and hyggenes, among others. Techniques for transforming fungi are well known inthe literature and have been described, for instance, by Beggs et al.((1978) Proc. Nat'l. Acad. Sci. USA 75: 1929-1933), Yelton et al.((1984) Proc. Natl. Acad. Sci. USA 81: 1740-1747), and Russell ((1983)Nature 301: 167-169). Procedures for transforming yeast are also wellknown (see, e.g., Beggs (1978) Nature (London), 275:104-109; and Hinnenet al. (1978) Proc. Natl. Acad. Sci. USA, 75:1929-1933. Transformationand infection methods for mammalian and other cells are described inBerger, Sambrook, and Ausubel, supra.

[0090] Once expressed, the FXR polypeptides and/or fusion proteins canbe purified, either partially or substantially to homogeneity, accordingto standard procedures of the art, such as, for example, ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Once purified, partially or to homogeneity asdesired, the polypeptides may then be used (e.g., in screening assaysfor modulators for gene expression or as immunogens for antibodyproduction).

[0091] One of skill in the art would recognize that after chemicalsynthesis, biological expression, or purification, the FXR polypeptidesand/or fusion proteins may possess a conformation substantiallydifferent than the native conformations of the constituent polypeptides.In this case, it may be necessary to denature and reduce the polypeptideand then to cause the polypeptide to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art (See, Debinski etal. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993)Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The protein is then refolded in a redox buffer containing oxidizedglutathione and L-arginine.

[0092] One of skill also would recognize that some modifications can bemade to the FXR polypeptides without diminishing their biologicalactivity. Such modifications can be made to facilitate the cloning,expression, or incorporation of the polypeptide into a fusion protein.Such modifications are well known to those of skill in the art andinclude, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

[0093] C. Chimeric FXR Polypeptides

[0094] The assays of the invention sometimes employ a chimeric FXRpolypeptide that has at least a ligand binding domain and a DNA bindingdomain. At least one of the ligand binding domain and the DNA bindingdomain of the chimeric receptors of the invention is substantiallyidentical to the corresponding domain of an FXR. These chimericreceptors are useful for many purposes. For example, one can use thechimeric receptors to identify additional ligands for the FXR and toidentify response elements that are responsive to FXR. The chimericreceptors are also useful in screening assays for identifying compoundsthat can modulate interactions between FXR and its ligands and/orresponse elements.

[0095] 1. DNA Binding Domains.

[0096] The chimeric receptors used in the assays of the inventioninclude those having a ligand binding domain that is at leastsubstantially identical to a ligand binding domain of an FXR, as well asthose that have a DNA binding domain that is not substantially identicalto a DNA binding domain of an FXR. For example, the DNA binding domainbinding domain can be about 90% or less identical to that of an FXR,more preferably about 75% or less, and most preferably about 60% or lessidentical. Often, the DNA binding domain is derived from a receptorother than a FXR. In a typical embodiment, the DNA binding domain is atleast substantially identical to a DNA binding domain from a nuclearhormone receptor or a steroid hormone receptor.

[0097] The modular nature of transcription regulators facilitates theconstruction of chimeric receptors that have domains that are derivedfrom different receptors (Green and Chambon (1986) Nature 324: 615-617).For example, DNA binding domains derived from steroid, thyroid, andretinoid hormone receptor are suitable for use in the chimeric receptorsof the invention. The DNA binding domains of receptors for steroid,thyroid, and retinoid hormones typically include two zinc finger units(Rhodes and Klug (February 1993) Scientific American, pp. 56-65). TheDNA binding domains of these receptors, are generally cysteine-richregions of about 65 amino acids that fold into two cysteine-rich “C4”type zinc fingers. The boundaries for many DNA binding domains have beenidentified and characterized for the steroid hormone superfamily. See,e.g., Giguere et al. (1986) Cell 46:645-652; Hollenberg et al. (1987)Cell 49:39-46; Green and Chambon (1987) Nature 325:74-78; Miesfield etal. (1987) Science 236:423-427; and Evans (1988) Science 240:889-895.

[0098] Examples of receptors from which one can derive DNA bindingdomains that are suitable for use in the chimeric receptors of theinvention include, for example, androgen receptors, estrogen receptors,glucocorticoid receptors, mineralcorticoid receptors, progesteronereceptors, retinoic acid receptors (including α, β (hap), and γ),thyroid hormone receptors (including α and β), the gene product of theavian erythroblastosis virus oncogene v-erbA (which is derived from acellular thyroid hormone receptor), vitamin D3 receptor, Drosophilaecdysone receptor (EcR), COUP transcription factor (also known as ear3)and its Drosophila homolog 7UP (svp), hepatocyte nuclear factor 4(HNF-4), Ad4BP, apolipoprotein AI regulatory protein-1 (ARP-1),peroxisome proliferator activated receptor (PPAR), Drosophila proteinknirps (kni), Drosophila protein ultraspiracle (usp; chorion factor 1),human estrogen receptor related genes 1 and 2 (err1 and err2), humanerbB related gene 2 (ear2), human NAK1/mouse nur/77 (N10)/rat NGFI-B;Drosophila protein embryonic gonad (egon), Drosophila knirps-relatedprotein (knr1), Drosophila protein tailless (tll), Drosophila20-O-ecdysone regulated protein E75, and Drosophila Dhr3. Some of theseand other suitable receptors are described in, for example, Evans, RM(1988) Science 240: 889-895; Gehrig, U. (1987) Trends Biochem. Sci. 12:399-402; Beato, M. (1989) Cell 56: 335-344; Laudet et al. (1992) EMBO J.11: 1003-1013.

[0099] In some embodiments, the chimeric receptors include a DNA bindingdomain from a DNA-binding polypeptide other than a nuclear receptor. Forexample, chimeric receptors that have the DNA binding domain of GAL4,which is a positive regulatory protein of yeast (Giniger et al. (1985)Cell 40: 767-774; Sadowski et al. (1992) Gene 118: 137-141) linked to aligand binding domain of an FXR polypeptide are provided. GAL4 DNAbinding domain-containing fusion proteins can be readily expressed bycloning a coding sequence for an FXR ligand binding domain into acommercially available expression vector that includes a GAL4 DNAbinding domain coding sequence under the control of a promoter (e.g.,pAS2-1 (CLONTECH Laboratories, Inc.). Another example of awell-characterized DNA binding domain for which expression vectors arecommercially available is that of LexA (pLexA, CLONTECH). The chimericreceptors can also include a nuclear localization sequence associatedwith the DNA binding domain (see, e.g., Silver et al. (1984) Proc.Nat'l. Acad. Sci. USA 81: 5951-5955 for a GAL4 nuclear localizationsequence).

[0100] The chimeric receptors can use an entire receptor molecule as aDNA binding domain, or can use portions of molecules that are capable ofbinding to nucleic acids, directly or indirectly. To identify such DNAbinding domains, one can perform assays such as an electrophoreticmobility shift assay (EMSA) (Scott et al. (1994) J. Biol. Chem. 269:19848-19858), in which a nucleic acid of interest is allowed toassociate with various fragments of a polypeptide to identify thosefragments that are capable of binding to the nucleic acid. Associationof a portion of the protein with the nucleic acid will result in aretardation of the electrophoretic mobility of the nucleic acid. Anothermethod by which one can identify DNA binding moieties that are suitablefor use as DNA binding domains is DNase I footprinting, which is wellknown to those of skill in the art.

[0101] The DNA binding domain can be either a polypeptide or a nucleicacid. Where the DNA binding domain is a nucleic acid, the nucleic acidwill be capable of specifically hybridizing to a target nucleic acidsite, such as a response element. Hybridization of the nucleic acid tothe target site will place the chimeric receptor in a position suitablefor activating or repressing expression of a gene that is linked to thetarget site. An example of an oligonucleotide being chemically linked toa protein by chemical coupling is found in Corey et al. (1989)Biochemistry 28: 8277-8286.

[0102] These chimeric receptors are useful, for example, in assays toidentify modulators of FXR transcriptional regulation activity asdescribed below.

[0103] 2. Ligand Binding Domains.

[0104] The assays of the invention typically use chimeric ornon-chimeric FXR receptors in which the ligand binding domain is atleast substantially identical to a ligand binding domain of an FXRpolypeptide.

[0105] 3. Production of Chimeric FXR Receptors

[0106] To form a chimeric receptor for use in the assay of theinvention, the ligand binding domain and the DNA binding domain arelinked together. Suitable methods of forming such linkages are known tothose of skill in the art. For a review of methods for constructingfusion proteins between receptor ligand binding domains and DNA bindingdomains, see, e.g., Mattioni et al. (1994) Methods in Cell Biology 43(PtA): 335-352. The linkage can be done using either recombinant orchemical methods. For example, a cysteine residue can be placed ateither end of a domain so that the domain can be linked to anotherdomain by, for example, a sulfide linkage. More typically, the ligandbinding domains and DNA binding domains are joined by linkers, which aretypically polypeptide sequences, such as poly glycine sequences ofbetween about 5 and 200 amino acids, with between about 10-100 aminoacids being typical. In some embodiments, proline residues areincorporated into the linker to prevent the formation of significantsecondary structural elements by the linker. Preferred linkers are oftenflexible amino acid subsequences which are synthesized as part of arecombinant fusion protein. In one embodiment, the flexible linker is anamino acid subsequence comprising a proline such as Gly(x)-Pro-Gly(x)where x is a number between about 3 and about 100. A linker can also bea single peptide bond, or one or more amino acid residues. In otherembodiments, a chemical linker is used to connect synthetically orrecombinantly produced ligand binding domain and DNA binding domainsubsequences. Such flexible linkers are known to persons of skill in theart. For example, poly(ethylene glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulfhydryl linkages, or heterofunctional linkages.

[0107] The chimeric receptors are conveniently produced by recombinantexpression in a host cell. Accordingly, the invention provides chimericnucleic acids that encode a fusion protein that includes a DNA bindingdomain and a ligand binding domain, at least one of which is at leastsubstantially identical to the corresponding domain of an FXR of theinvention. In some embodiments, the chimeric nucleic acid will alsoencode a linker region that provides a link between the two domains.Techniques for making such chimeric nucleic acids are known to those ofskilled in the art. For example, recombinant methods can be used (see,e.g., Berger and Sambrook, both supra.). Alternatively, the nucleic acidencoding the chimeric receptors can be synthesized chemically.

[0108] To obtain expression of a chimeric receptor, a nucleic acid thatencodes the chimeric receptor is generally placed under the control of apromoter and other control elements that can drive expression of thechimeric gene in a desired host cell. Accordingly, the invention alsoprovides expression cassettes in which a promoter and/or other controlelements are operably linked to a polynucleotide that encodes a chimericreceptor. Suitable promoters, other control sequences, and expressionvectors are described above.

[0109] D. Assays for FXR Ligands and Response Elements

[0110] Also provided by the invention are methods for identifyingcorepressors, coactivators, and additional ligands that interact withFXR. Also provided are methods of identifying response elements that aremediated by transcription complexes that include FXR. These componentsare useful in the screening assays of the invention.

[0111] 1. Identification of Ligands for FXR

[0112] Many of the screening assays of the invention utilize ligands forFXR. Accordingly, the invention provides methods for identifyingadditional ligands for FXR. The identification of previously unknown FXRligands is of particular interest not only for the knowledge obtainedregarding the regulation of cholesterol metabolism, but also foridentifying new compounds that can modulate FXR-mediated regulation ofgenes that are involved in cholesterol metabolism.

[0113] Candidate ligands include not only bile acids and relatedcompounds, but also transcription factors, coactivators, andcorepressors with which FXR might interact. These potential ligands caninclude other receptor polypeptides (e.g., RXR, coactivators, and thelike), which comprise the cellular machinery for regulation of geneexpression. For example, nuclear hormone receptors often interact withtranscriptional coactivators. Thus, the invention also provides methodsof identifying coactivators, corepressors and other molecules thatinteract with FXRs. These assay methods can involve introducing acoactivator or a corepressor that is a candidate ligand for FXR into ahost cell that contains a chimeric FXR and reporter plasmid. Thecoactivator can be introduced by means of an expression construct; thisexpression construct can be present on the same or a different vectorthan the expression construct for the chimeric receptor.

[0114] Ligands for FXR can be identified using the methods describedabove for screening to identify compounds that modulate FXR interactionswith the ligands. Instead of including a compound that potentiallymodulates the interaction, the assays are conducted using a potentialFXR ligand. Both polypeptide-based and cell-based assays can be used.

[0115] 2. Identification of Response Elements for TranscriptionComplexes that Include FXR

[0116] The present invention provides methods for obtaining responseelements that are responsive to transcription complexes that includeFXR. Through use of such methods, one can identify additional genes forwhich expression is modulated by the FXRs. The methods typically involvecontacting a putative response element with a polypeptide that includesa FXR DNA binding domain (see, e.g., Ausubel et al., supra.). Bothcell-based and biochemical methods are provided. In presently preferredembodiments of the assays for identification of FXR response elements, aFXR receptor, or FXR DNA binding domain is used. The ligand bindingdomain is preferably one for which an appropriate ligand is available.Suitable chimeric receptors are described above.

[0117] Also provided are methods of identifying response elements towhich FXR does not directly bind. For example, FXR can form atranscription regulatory complex with one or more other coactivatorsand/or corepressors. One or more of these other molecules can actuallybind to the response element. For example, FXR can bind to CPF, which inturn binds to a response element located upstream of the cyp7a gene.

[0118] In some embodiments, standard gel shift assays are performed toidentify polynucleotides that can bind to a FXR DNA binding domain.These assays are performed by incubating a polypeptide that includes aFXR DNA binding domain, either as a purified protein or a complexmixture of proteins) with a labeled DNA fragment that contains theputative FXR binding site. Reaction products are analyzed on anondenaturing polyacrylamide gel. To determine the specificity of thebinding, one can perform competition experiments using polynucleotidesthat include a FXR binding site, or unrelated DNA sequences. Kits forperforming gel shift assay include, for example, Gel Shift Assay Systems(Promega, Madison Wis., Part No. TB110).

[0119] Another in vitro assay for identifying FXR response elements isthe binding site selection method (see, e.g., U.S. Pat. No. 5,582,981).In this method, a library of oligonucleotides having a randomizednucleotide sequence of about 18 nucleotides flanked by two knownnucleotide sequences of sufficient length to allow hybridization to PCRprimers that are complementary to these regions. The oligonucleotidesare end-labeled (e.g., with γ-³²P) and contacted with a FXR DNA bindingdomain polypeptide. A low stringency gel shift experiment is performed.PCR amplification is then carried out on those oligonucleotides to whichthe FXR DNA binding domain bound, as evidenced by retardation in the gelshift electrophoresis. Preferably, the selection and amplificationprocess is repeated at least twice more using the amplified fragments.

[0120] In vivo assays for FXR response elements are also provided. Thein vivo assays are particularly suitable for confirming results obtainedin an in vitro assay. Cells are provided which contain a reporterconstruct that contains the putative response element in a positionrelative to a promoter at which binding of an FXR polypeptide canincrease or decrease expression of an operably linked gene. The putativeresponse element can be, for example, a member of a library ofpolynucleotide fragments. The chimeric receptor and the reporterconstructs are introduced into a host cell. Suitable host cells aredescribed in, for example, U.S. Pat. No. 5,071,773. The host cells thatcontain the reporter plasmid construct and the chimeric receptor aregrown in the presence of the ligand for the ligand binding domain usedin the chimeric receptor. Those cells in which expression of thereporter gene in the presence of the ligand is greater or less than theexpression in the absence of the ligand contain a reporter constructthat includes a putative response element for an FXR. The responseelements can be isolated from these cells by, for example, plasmidrecovery, PCR amplification, or other methods known to those of skill inthe art. Upon isolation, the response elements can be characterized(e.g., by sequencing) and used to identify additional genes for whichexpression is influenced by FXRs.

[0121] E. Compositions, Kits and Integrated Systems

[0122] The invention provides compositions, kits and integrated systemsfor practicing the assays described herein. For example, the inventionprovides an assay system that includes an FXR polypeptide and a ligandfor FXR. Also provided are assay systems for cell-based screening toidentify FXR-modulating compounds. Such systems typically include anexpression vector for a full-length or chimeric FXR polypeptide, avector that contains an appropriate reporter gene under the control of atranscription complex that includes FXR, and a suitable host cell isprovided by the present invention. Ligands that bind to the ligandbinding domain of FXR can also be included in the assay compositions, ascan modulators of FXR activity.

[0123] The invention also provides kits for practicing the FXR assaymethods noted above. The kits can include any of the compositions notedabove, and optionally further include additional components such aswritten instructions to practice a high-throughput method of assayingfor FXR activity, or screening for an inhibitor or activator of FXRactivity, one or more containers or compartments (e.g., to holdreagents, nucleic acids, or the like), and a control FXR activitymodulator.

[0124] The invention also provides integrated systems forhigh-throughput screening of potential FXR modulators for an effect onbinding of FXR to ligands for FXR. In other some systems, the modulationof expression of genes that are under the control of FXR, such as thecyp7a gene that modulates cholesterol metabolism, is tested. The systemstypically include a robotic armature which transfers fluid from a sourceto a destination, a controller which controls the robotic armature, alabel detector, a data storage unit which records label detection, andan assay component such as a microtiter dish comprising a well having areaction mixture or a substrate.

[0125] F. Pharmaceutical Compositions and Methods for TreatingCholesterol-related Disorders

[0126] FXR-mediated disorders and conditions, such as atherosclerosis,cardiovascular disorders, lipid disorders and hypercholesterolemia, canbe treated with therapeutic agent(s) identified using the methodsdescribed herein. The candidate therapeutic agent is typically preparedas a pharmaceutical composition and is administered to a subjectsuffering from a FXR-mediated disorder or condition. Preferably, thecandidate therapeutic agents will, upon administration to the subject,cause total cholesterol levels to decrease about 10%, more preferably adecrease of about 20%, and most preferably a decrease of about 25-45%.

[0127] 1. Pharmaceutical Compositions

[0128] Accordingly, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient and a candidate therapeutic agent. Pharmaceutically acceptablecarriers can be either solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. A solid carrier can be one or more substanceswhich may also act as diluents, flavoring agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial.

[0129] In powders, the carrier is a finely divided solid which is in amixture with the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

[0130] The powders and tablets preferably contain from 5% or 10% to 70%of the active compound. Suitable carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

[0131] For preparing suppositories, a low melting wax, such as a mixtureof fatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

[0132] Liquid form preparations include solutions, suspensions, andemulsions, for example, water or water/propylene glycol solutions. Forparenteral injection, liquid preparations can be formulated in solutionin aqueous polyethylene glycol solution.

[0133] Aqueous solutions suitable for oral use can be prepared bydissolving the active component in water and adding suitable colorants,flavors, stabilizers, and thickening agents as desired. Aqueoussuspensions suitable for oral use can be made by dispersing the finelydivided active component in water with viscous material, such as naturalor synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

[0134] Also included are solid form preparations which are intended tobe converted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

[0135] The pharmaceutical preparation is preferably in unit dosage form.In such form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

[0136] The quantity of active component in a unit dose preparation maybe varied or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100mg according to the particular application and the potency of the activecomponent. The composition can, if desired, also contain othercompatible therapeutic agents.

[0137] 2. Treatment Regime Using Candidate Therapeutic Agents

[0138] The present invention also provides methods of modulating FXRactivity in a cell. In this aspect, a cell is contacted with anFXR-modulating amount of a compound or composition above. AnFXR-modulating amount can be readily determined using the assaysdescribed briefly above, or alternatively, using the assays in theExamples below. Candidate therapeutic agents are especially useful inthe treatment of hypercholesterolemia.

[0139] In another aspect, the present invention provides methods oftreating conditions modulated by FXR in a host animal, by administeringto the host an effective amount of a compound or composition providedabove. In therapeutic applications, the compounds of the presentinvention can be prepared and administered in a wide variety of oral andparenteral dosage forms. Thus, the compounds of the present inventioncan be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present invention can be administered transdermally.

[0140] A variety of conditions are modulated, at least in part, by FXR,including hypercholesterolemia or other conditions associated withabnormal cholesterol or lipid homeostasis such as atherosclerosis, lipiddisorders and cardiovascular disorders. The compounds utilized in thepharmaceutical method of the invention are administered at the initialdosage of about 0.001 mg/kg to about 100 mg/kg daily. A daily dose rangeof about 0.1 mg/kg to about 10 mg/kg is preferred. The dosages, however,may be varied depending upon the requirements of the patient, theseverity of the condition being treated, and the compound beingemployed. Determination of the proper dosage for a particular situationis within the skill of the practitioner. Generally, treatment isinitiated with smaller dosages which are less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under circumstances is reached. Forconvenience, the total daily dosage may be divided and administered inportions during the day, if desired.

[0141] Typically, the host or subject in each of these methods is human,although other animals can also benefit from the foregoing treatments.

EXAMPLES

[0142] The following examples are offered to illustrate, but not tolimit the present invention. Some of these experiments are described inMakishima et al. (1999) Science 284: 1362-1365, which was publishedafter the priority date of the instant application.

Example I CDCA Specifically Transactivates FXR

[0143] This Example demonstrates that the primary bile acidchenodeoxycholic acid (CDCA) specifically activates the gene expressionregulatory activity of FXR. The experiment was conducted using fusionpolypeptides in which a ligand binding domain of FXR and other nuclearreceptors were independently linked to the DNA binding domain (DBD) ofGal4. These in-frame fusions of the Gal4 DNA-binding domain (Gal4 DBD,pM3 expression vector) and the ligand-binding domains (LBDs) of FXR,HNF4α, LXRα, RXRα and LXRβ nuclear receptors were used to determine thespecificity of FXR transactivation by chenodeoxycholic acid (CDCA).

[0144] HepG2 cells were transiently transfected with a reporter plasmidin which a luciferase-encoding gene was under the control of a pGL3 5×GAL4 response element (which consists of five GAL4 binding elements)(0.25 μg/1.5×10⁵ cells). The cells were also transfected with the emptyexpression vector pM3 or the pM3-LBD fusion expression plasmids (0.25μg/1.5×10⁵ cells) as indicated. Luciferase reporter activity wasmeasured after treating cells with or without 50 μM CDCA. For eachfusion plasmid, fold activation is a ratio of luciferase activity(normalized by β-galactosidase activity) between treated and untreatedcells. The FXR fusion construct was the sole receptor transactivated byCDCA (see, FIG. 1A).

[0145] This example further illustrates that CDCA-mediatedtransactivation of FXR is dose-dependent. HepG2 cells were transientlytransfected with the pGL3 3× FXRE (three FXR response elements,AGGTCAATGACCT) luciferase reporter plasmid (0.25 μg/1.5×10⁵ cells), plusempty expression vector (pcDNA3) or a plasmid expressing full-length FXR(pFXR) (0.15 μg/1.5×10⁵ cells). Luciferase reporter activity wasmeasured after treating cells with or without CDCA (concentrations asindicated). Fold activation was calculated as the ratio of luciferaseactivity (normalized by β-galactosidase activity) between treated anduntreated cells. A dose-dependent transactivation (IC₅₀ 10 μM CDCA) ofthe reporter gene by FXR was observed (see, FIG. 1B).

Example II FXR Suppresses CYP7 Expression

[0146] This example illustrates that CDCA-mediates FXR-specificsuppression of CYP7 expression. Full-length nuclear receptors FXR, CPF,HNF4α, LXRα and RXRα were assayed to determine their ability to suppressCYP7 expression in the presence of 10 μM CDCA. In this experiment, aluciferase reporter plasmid under the control of the cyp7a promoter(−718 to +14, where +1 designates the transcriptional start site)(pGL3-CYP7) was used. HepG2 cells were transiently transfected with thepGL3-CYP7 luciferase reporter (0.25 μg/1.5×10⁵ cells) plus emptyexpression vector (pcDNA3) or plasmids expressing one of the indicatednuclear receptors (0.25 μg/1.5×10⁵ cells). Luciferase reporter activitywas measured after treating cells with or without 10 μM CDCA. The ratioof luciferase activity (normalized by β-galactosidase activity) betweentreated and untreated pcDNA3-transfected cells was set at 100 percentCYP7 expression. The percent CYP7 expression for the nuclear receptorswas normalized to that of pcDNA3. Only FXR suppressed CYP7 expressionunder these conditions (see, FIG. 2A).

[0147] This example further illustrates the dose response profile ofCDCA-mediated suppression of CYP7 expression by FXR. HepG2 cells weretransiently transfected with the pGL3-CYP7 luciferase reporter plasmid(0.25 μg/1.5×10⁵ cells), plus empty expression vector (pcDNA3) or aplasmid expressing full-length FXR (pFXR) (0.15 μg/1.5×10⁵ cells).Luciferase reporter activity was measured after treating cells with orwithout CDCA (concentrations as indicated). The presence of FXR alonesuppresses CYP7 expression, and this suppression is enhanced with theaddition of CDCA (see, FIG. 2B).

Example III FXR Suppression Specific to CYP7

[0148] This example illustrates that CDCA-mediated FXR suppression isspecific for CYP7. FXR does not suppress expression of the low densitylipoprotein (LDL) receptor. This experiment was conducted using a secondluciferase reporter plasmid, named p-6500, in which the luciferase geneis under the control of a 6.5 kilobase pair (kb) promoter region of theLDL receptor. HepG2 cells were transiently transfected with pGL3-CYP7 orp-6500. Cells were also transfected with either empty expression vector(pcDNA3) or a plasmid that expresses full-length FXR (pFXR) (all at 0.25μg/1.5×10⁵ cells). Luciferase reporter activity was measured aftertreating cells with or without 10 μM CDCA. The ratio of luciferaseactivity (normalized by β-galactosidase activity) between treated anduntreated pcDNA3-transfected cells was set at 100 percent CYP7expression. No FXR/CDCA-mediated suppression of the p-6500 reporter wasobserved (see, FIG. 3A).

[0149] This example further illustrates that CYP7 suppression is lostupon titrating away FXR. HepG2 cells were transiently transfected withpGL3-CYP7 luciferase reporter plasmid (50 ng/1.5×10⁵ cells), plus anempty expression vector (pcDNA3) or a plasmid expressing full-length FXR(pFXR) (0.25 μg/1.5×10⁵ cells). Cells were also transfected with acompetitor plasmid, either pBSKS or pGL3 3× FXRE Aluc (0.5 μg/1.5×10⁵cells). Luciferase reporter activity was measured after treating cellswith or without 10 μM CDCA. For each competitor, the ratio of luciferaseactivity (normalized by β-galactosidase activity) between treated anduntreated pcDNA3-transfected cells was set at 100 percent CYP7expression. Loss of CYP7 suppression was observed when ten-fold excesspGL3 3× FXR Aluc was cotransfected with the CYP7 reporter gene (see,FIG. 3B). This result strongly suggests that FXR is a component of thebile acid-mediated suppression of CYP7.

[0150] This example further illustrates that FXR mutants do not suppressCYP7 expression. FXR mutants were created and assayed to determinewhether they are able to suppress CYP7 in a CDCA-dependent manner. HepG2cells were transiently transfected with the pGL3-CYP7 luciferasereporter plasmid (0.25 μg/1.5×10⁵ cells), plus empty expression vector(pcDNA3) or a plasmid expressing full-length wild-type FXR (pFXR) ormutant (DNA-binding domain [DBD], ligand-binding domain [pLBD], and AF2domain truncation [dAF2]) FXR (0.25 μg/1.5×10⁵ cells). Luciferasereporter activity was measured after treating cells with or without 10μM CDCA. The ratio of luciferase activity (normalized by β-galactosidaseactivity) between treated and untreated pcDNA3-transfected cells was setat 100 percent CYP7 expression. None of the FXR derivatives had anyeffect upon CYP7 suppression (see, FIG. 3C).

Example IV Bile Acids and FXR Repress Cyp7A Promoter

[0151] This example demonstrates that bile acids and FXR repressexpression from the promoter for the cholesterol 7α-hydroxylase (Cyp7A)gene, which is the rate-limiting enzyme in cholesterol metabolism. Boththe expression of Cyp7A protein and mRNA (FIG. 4A in ahepatocyte-derived cell line were analyzed. HepG2 cells were seeded atan initial density of 1×10⁶ cells/well in a 6-well plate, and allowed togrow for approximately 24 hours at 37° C., 5% CO₂ in DMEM-F12 medium(Mediatech) supplemented with 10% fetal calf serum. Once the cellculture became confluent, the medium was replaced with DMEM-F12 withoutserum, and supplemented with primary or conjugated bile acids at a finalconcentration of 50 μM. The cultures were then incubated at 37° C., 5%CO₂ for 24 hours.

[0152] An immunoblot analysis using an antibody to Cyp7a was performedon lysates from human HepG2 cells after treatment with bile acids.Quantitative RT-PCR analysis of CYP7A mRNA in HepG2 cells was performedas follows. Total RNA was prepared from HepG2 cells using Tri Reagent(Molecular Research Center, Inc.), according to the recommendations ofthe manufacturer. Following extraction with Tri Reagent andprecipitation with ethanol, the RNA was resuspended in DEPC-treatedwater and stored at −80° C. prior to analysis. The following primerswere used for amplification and analysis of human CYP7 mRNA by RT-PCR:CYP7⁻⁷⁸: 5′-tgatttgggggattgctata-3′, CYP7⁻¹⁷⁸:5′-catacctgggctgtgctct-3′, and CYP7⁻¹³²(FAM):5′-(6-FAM)tggttcacccgtttgccttctcct(TAMRA)-3′. As a control, a primer setfor detection of human GAPDH mRNA was used; this set is commerciallyavailable and was obtained from Applied Biosystems/Perkin Elmer, Inc.

[0153] Amplification of specific target mRNAs was carried out by reversetranscription followed by polymerase chain reaction using the TaqMan OneStep Gold RT-PCR Kit (Applied Biosystems/Perkin Elmer, Inc.). Eachprimer set included a pair of amplification primers and the fluorogenicprobe primer as indicated, and were present in the reaction at a finalconcentration of 100 nM/primer. One μg of total RNA isolated from thevarious culture conditions was used as template in individual RT-PCRreactions.

[0154] Analysis of GAPDH mRNA and CYP7A mRNA were carried out intriplicate in parallel reactions. Reactions were carried out using theABI Prism 7700 Sequence Detector (Perkin Elmer, Inc.) Reversetranscription was allowed to proceed for 30 minutes at 48° C., followedby amplification of the cDNA by 40 cycles of PCR, each consisting of amelting step (95° C. for 15 seconds) and a combined annealing-extensionstep (60° C. for 1 minute). During PCR, the cycle-to-cycle changes influorescence due to template amplification were monitored, allowing theamount of amplified product to be determined, and the initial templateconcentration to be calculated. These values were used to compare thesteady-state levels of accumulated CYP7A and GAPDH mRNA in HepG2 cellsgrown in the presence and absence of bile acids (see, FIG. 4A).

[0155] Also examined was the effect of bile acids on coactivatorrecruitment to FXR. A mammalian two-hybrid assay was used to demonstratethat FXR interacts with the coactivator SRC-1. In this experiment, aplasmid expressing a GAL4 DNA-binding domain-SRC-1 fusion protein and aplasmid expressing an FXR ligand-binding domain-VP-16 fusion proteinwere used. HepG2 cells were transiently transfected with the pG5luciferase reporter plasmid (a luciferase gene under the control of fiveGAL4 binding sites) (0.25 μg/1.5×10⁵ cells) and the plasmids expressingthe two fusion proteins. Luciferase reporter activity was measured aftertreating cells with or without the panel of bile acids (concentrationsas indicated) (FIG. 4B). Relative luciferase activity is a ratio ofluciferase activity (normalized by β-galactosidase activity) betweentreated and untreated cells.

[0156] The ability of different bile acids and derivatives to activateFXR-mediated transactivation was also examined. HepG2 cells weretransiently transfected with the pGL3 3× FXRE luciferase reporterplasmid (0.25 μg/1.5×10⁵ cells) and a plasmid expressing full-length FXR(pFXR) (0.15 μg/1.5×10⁵ cells). Luciferase reporter activity wasmeasured after treating cells with or without the panel of bile acids(concentrations as indicated). Fold activation is a ratio of luciferaseactivity (normalized by β-galactosidase activity) of treated anduntreated cells. Treatment with CDCA, followed by deoxycholic acid (DCA)and glycochenodeoxycholic acid (GCDCA), had the greatest induction ofluciferase reporter activity (FIG. 4C).

[0157] The effect of different bile acids and derivatives onFXR-mediated suppression of CYP7 expression was also studied. HepG2cells were transiently transfected with the pGL3-CYP7 luciferasereporter plasmid (0.25 μg/1.5×10⁵ cells) and a plasmid expressingfull-length FXR (pFXR) (0.15 μg/1.5×10⁵ cells). Luciferase reporteractivity was measured after treating cells with or without the panel ofbile acids (concentrations as indicated). Luciferase reporter activityfor untreated cells was defined as 100% percent CYP7 expression. Thehighest level of suppression of CYP7 expression was observed with CDCAtreatment followed by DCA (see, FIG. 4D).

[0158] CDCA treatment resulted in the largest induction of luciferaseactivity (see, FIGS. 4B and 4C), and also the largest repression ofCYP7a expression (FIG. 4D). The rank order of bile acids in theseactivity-measuring experiments was the same as that for repressingendogenous expression of human Cyp7a protein and mRNA (FIG. 4A)

Example V High-Throughput in vitro Biochemical Assays

[0159] This Example describes three different high throughput in vitroassay that are useful for screening to identify compounds that canmodulate binding of FXR ligands to the FXR ligand binding domain. Theability of different bile acids and derivatives to affect the binding ofa labeled sensor peptide that is derived from the coactivator SRC-1 toan FXR ligand binding domain was tested.

[0160] Fluorescence Polarization

[0161] Fluorescence polarization was used to study the effect ofdifferent bile acids on the ability of the FXR LBD to bind a sensorpeptide. The assay reagents were as follows:

[0162] Reagents:

[0163] Sensor: Rhodamine-labeled ILRKLLQE peptide (final conc.=1-5 nM).It is noted that the Rhodamine-labeled peptide comprises, at a minimum,the following sequence LXXLLXX, wherein X is any amino acid. Additionalamino acids can be added to both the N-terminus and the C-terminus ofthis core peptide. In preferred embodiments, the peptide is 8 aminoacids in length and, more preferable, about 11 amino acids in length.

[0164] Receptor: Glutathione-S-transferase/FXR ligand binding domainfusion protein (final conc.=100-200 nM).

[0165] Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6

[0166] Protocol:

[0167] 1. Add 90 microliters of Sensor/Receptor mixture to each well ofa 96-well microtiter plate.

[0168] 2. Add 10 microliters of test compound per well.

[0169] 3. Shake 5 minutes and within 5 minutes determine the amount offluorescence polarization by using a Fluorolite FPM-2 FluorescencePolarization Microtiter System (Dynatech Laboratories, Inc.

[0170] Ten ng/μL of GST-FXR fusion protein was mixed with arhodamine-labeled peptide comprising LXXLLXX, wherein X is any aminoacid, and the panel of bile acids (concentrations as indicated).Fluorescence polarization was read after a room temperature incubationand brief shaking. Change in millipolarization (mP) units is thedifference treated and untreated samples. The high change in mP unitsdemonstrates that the labeled peptide binds to GST-FXR in aCDCA-dependent manner (see, FIG. 5A).

[0171] Fluorescence Resonance Energy Transfer

[0172]FIG. 5B shows the results of an experiment in which fluorescenceresonance energy transfer (FRET; also referred to as HTRF) was used tostudy the effect of different bile acids on the ability of FXR to bindthe coactivator SRC-1. The top panel shows that FXR-SRC-1 binding isstimulated by bile acids. The bottom panel of FIG. 5B shows that thebinding of the LXRα receptor control is affected not by the bile acids,but rather by the LXRα ligand 24,25 epoxycholesterol.

[0173] ELISA

[0174] An enzyme-linked immunosorbent assay (ELISA) was used to studythe effect of bile acids on ligand-induced conformational changes ofFXR. A peptide sensor was used. The results, which are shown in FIG. 5C,demonstrate that increasing concentrations of bile acids (in particular,CDCA) result in an increase in the amount of peptide sensor that isbound to the FXR ligand binding domain. The binding of the sensorpeptide to the LXRα control (bottom panel), in contrast, was notaffected by the bile acids. Only the LXRα ligand 24,25 epoxycholesterolresulted in increased binding of the sensor peptide to LXRα.

[0175] From the foregoing examples, it is readily apparent that theorphan nuclear receptor FXR is involved in the bile acid-dependentsuppression of the human cyp7 gene. The primary bile acid CDCA(chenodeoxycholic acid) specifically transactivates FXR and does so in adose-dependent manner (IC₅₀=10 M). Overexpression of FXR was found tosuppress cyp7 expression and the suppression was enhanced in thepresence of CDCA. This enhanced suppression is specifically mediated byFXR and seems to be specific to the cyp7 promoter. By using a panel ofbile acids, it was demonstrated that the bile acid-mediated FXRactivation correlates with bile acid-mediated human cyp7 suppression.Moreover, by using an in vitro biochemical peptide sensor assay and thecell-based mammalian two-hybrid assay, it was demonstrated that when FXRis bound by CDCA, the activated complex is able to recruit SRC-1 to thecomplex. In summary, the results strongly suggest that FXR functions asa bile acid receptor/sensor that mediates cyp7 expression in a bile-aciddependent manner.

[0176] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

What is claimed is:
 1. A method of prescreening to identify a candidatetherapeutic compound suitable for testing for ability to modulatecholesterol metabolism in a cell, said method comprising: providing areaction mixture that comprises: a) a polypeptide that comprises aligand binding domain of an FXR; b) a ligand for FXR; and c) a testcompound; and determining whether the amount of binding of the FXRligand binding domain to the ligand for FXR is increased or decreased inthe presence of the test compound compared to the amount of binding inthe absence of the test compound; wherein a test compound that causes anincrease or decrease in binding is a candidate therapeutic agent formodulation of cholesterol metabolism.
 2. The method of claim 1, whereinthe method further comprises administering the candidate therapeuticagent to a cell to determine whether the candidate therapeutic agentmodulates cholesterol metabolism in the cell.
 3. The method of claim 2,wherein the cell is in a mammal.
 4. The method of claim 3, wherein thecompound is administered to the cell by administration to the mammal. 5.The method of claim 1, wherein the ligand for FXR is a peptide sensor.6. The method of claim 5, wherein the peptide sensor is derived from acoactivator or corepressor.
 7. The method of claim 6, wherein thecoactivator is SRC-1.
 8. The method of claim 7, wherein the peptidesensor comprises an amino acid sequence LXXLL, wherein L is leucine andX is any amino acid.
 9. The method of claim 5, wherein the peptidesensor comprises a detectable label.
 10. The method of claim 1, whereinthe ligand is a bile acid or bile acid derivative.
 11. The method ofclaim 10, wherein said bile acid is a member selected from the groupconsisting of CDCA, GCDCA, TCDCA, GCA, TCA, DCA and CA.
 12. The methodof claim 11, wherein said bile acid is CDCA.
 13. The method of claim 1,wherein the ligand is a coactivator or a corepressor.
 14. The method ofclaim 13, wherein the coactivator is SRC-1.
 15. The method of claim 1,wherein the amount of binding is determined using a FRET assay.
 16. Themethod of claim 1, wherein the amount of binding is determined using afluorescence polarization assay.
 17. The method of claim 1, wherein theamount of binding is determined using ELISA.
 18. The method of claim 1,wherein the amount of binding is determined using a direct bindingassay.
 19. A method for increasing cholesterol metabolism in a cell,said method comprising contacting said cell with a compound thatmodulates the binding of FXR to a ligand of FXR.
 20. The method inaccordance with claim 19, wherein said ligand of FXR is a bile acid. 21.The method in accordance with claim 20, wherein said bile acid is amember selected from the group consisting of CDCA, GCDCA, TCDCA, GCA,TCA, DCA and CA.
 22. The method in accordance with claim 21, whereinsaid bile acid is CDCA.
 23. The method in accordance with claim 19,wherein said ligand of FXR is RXR.
 24. The method in accordance withclaim 19, wherein said ligand of FXR is a coactivator.
 25. The method inaccordance with claim 19, wherein said ligand of FXR is a corepressor.26. The method in accordance with claim 19, wherein said compound is anantibody that binds to FXR.
 27. The method in accordance with claim 19,wherein said cell is a mammalian cell.
 28. The method in accordance withclaim 19, wherein said cell is in a mammal.
 29. The method in accordancewith claim 19, wherein said compound modulates binding of atranscription complex that comprises FXR to a response element.
 30. Themethod in accordance with claim 29, wherein the response element isderived from a region upstream of a cyp7 gene.
 31. A method for reducingcholesterol levels in a mammal, said method comprising administering tosaid mammal a compound that modulates the binding of FXR to a ligand ofFXR.
 32. The method in accordance with claim 31, wherein said mammal isa human.
 33. A method of screening to identify a compound that modulatescholesterol metabolism in a cell, said method comprising: contacting acell with a test compound, wherein said cell comprises: a) apolynucleotide that encodes a polypeptide comprising: 1) a DNA bindingdomain of a receptor which binds to DNA; and 2) a ligand binding domainthat is substantially identical to a ligand binding domain of a FXR; b)a ligand for FXR; and c) a reporter gene construct which comprises aresponse element to which said DNA binding domain can bind, wherein saidresponse element is operably linked to a promoter that is operative inthe cell and said promoter is operably linked to a reporter gene; anddetermining whether said reporter gene is expressed at a higher or lowerlevel in the presence of said test compound compared to said reportergene expression level in the absence of said test compound, wherein atest compound that causes an increase or decrease in reporter geneexpression can modulate cholesterol metabolism in a cell.
 34. The methodin accordance with claim 33, wherein said polypeptide is FXR.
 35. Themethod in accordance with claim 34, wherein said FXR is a human FXR. 36.The method in accordance with claim 34, wherein increased expression ofsaid reporter gene in the presence of said test compound is indicativeof increased cholesterol metabolism.
 37. The method in accordance withclaim 33, wherein said cell also comprises an RXR polypeptide.
 38. Themethod in accordance with claim 37, wherein said RXR polypeptide is ahuman RXR polypeptide.
 39. The method in accordance with claim 33,wherein said FXR is a human FXR.
 40. The method in accordance with claim33, wherein said DNA binding domain is derived from a receptor selectedfrom the group consisting of an estrogen receptor, a progesteronereceptor, a glucocorticoid receptor, an androgen receptor, amineralcorticoid receptor, a vitamin D receptor, a retinoid receptor,and a thyroid hormone receptor.
 41. The method in accordance with claim33, wherein DNA binding domain is a GAL4 DNA binding domain.
 42. Themethod in accordance with claim 41, wherein increased expression of saidreporter gene in the presence of said test compound is indicative ofincreased ability of the compound to stimulate cholesterol metabolism.43. The method in accordance with claim 33, wherein said DNA bindingdomain is substantially identical to a DNA binding domain of a FXR. 44.The method in accordance with claim 43, wherein said response element isderived from an upstream region of a cyp7 gene.
 45. The method inaccordance with claim 44, wherein decreased expression of said reportergene in the presence of said test compound is indicative of increasedability of the compound to stimulate cholesterol metabolism.
 46. Themethod in accordance with claim 33, wherein said bile acid is a memberselected from the group consisting of CDCA, GCDCA, TCDCA, GCA, TCA, DCAand CA.
 47. The method in accordance with claim 46, wherein said bileacid is CDCA.